Invasive adenylate cyclase toxin of Bordetella pertussis

Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation.     

A protein activator for the adenylate cyclase of Bordetella pertussis.

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase is 100-fold higher in organisms grown on blood agar than in those grown in synthetic medium. This increase in activity is due to in vivo activation of the enzyme by a factor present in erythrocytes. Activation also occurs in killed or disrupted organisms. The activator can be separated from heme proteins and has been purified approximately 100-fold from erythrocytes, yielding material of approximately 105,000 daltons. It is sensitive to trypsin and alpha-chymotrypsin and exhibits considerable heat stability. Activation of cyclase in intact B. pertussis organisms exhibits a lag of 3 to 4 min and is not reversed by washing. Response to the activator decreases with increasing purification of the adenylate cyclase and is absent in the pure enzyme. The activation does not appear to be proteolytic and does not appear to change access to the substrate, ATP. The activator has no effect on a number of eukaryotic cyclases. We conclude that this is a new type of activation and that the activator differs from all those previously described.     

Assay of calmodulin with Bordetella pertussis adenylate cyclase

Low levels of the calcium-dependent regulator protein, calmodulin, may be measured utilizing membranes prepared from Bordetella pertussis which contain and adenylate cyclase which is activated by this protein. The activation is dose dependent and tissue levels of calmodulin can be determined over a range from 2 pg to 100 ng with good reliability. We demonstrate how this bioassay may be employed to measure the levels of calmodulin in a variety of protein and cellular preparations.     

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

Effect of methyl-cyclodextrin on adenylate cyclase activity of Bordetella pertussis

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase (AC) decreased during decelerating growth phase in a Stainer-Scholte medium. Neither proteolytic activity nor virulence variation (phase variation; antigenic modulation) appears to be responsible for the observed activity fall. The addition of methyl--cyclo-dextrin enhances AC activity and prevents the inhibition of AC activity by fatty acids. Cyclodextrin could entrap inhibitors increasing in this way the AC activity. These results show that the inclusion of cyclodextrin in the culture medium increases the AC activity.D.F. Hozbor and O.M. Yantorno are with the Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, (1900) La Plata, Argentina. A. Samo is with the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Calmodulin inhibits entry of Bordetella pertussis adenylate cyclase into animal cells

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Recently, Confer and Eaton [Confer, D., & Eaton, J. (1982) Science (Washington, D.C.) 217, 948-950], as well as Hanski and Farfel [Hanski, E., & Farfel, Z. (1985) J. Biol. Chem. 290, 5526-5536], have shown that crude extracts from B. pertussis containing adenylate cyclase activity cause elevations in intracellular cAMP when incubated with human neutrophils or lymphocytes. These investigators proposed that the bacterial enzyme enters animal cells and catalyzes the formation of cAMP from intracellular ATP. In this study, B. pertussis adenylate cyclase was purified to remove contaminating islet activating protein and examined for its effects on intracellular cAMP levels of human erythrocytes and N1E-115 mouse neuroblastoma cells. In both cases, the enzyme catalyzed the formation of intracellular cAMP. Addition of calmodulin to the adenylate cyclase preparations completely inhibited formation of intracellular cAMP catalyzed by the bacterial enzyme, indicating that cAMP was not synthesized extracellularly and then taken up by the cells. These experiments illustrate that the bacterial enzyme does enter animal cells and that the enzyme-calmodulin complex does not.     

Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the bacterium responsible for whooping cough, releases a soluble, calmodulin-sensitive adenylate cyclase into its culture medium. B. pertussis mutants deficient in this enzyme are avirulent, indicating that the adenylate cyclase contributes to the pathogenesis of the disease. It has been proposed that B. pertussis adenylate cyclase may enter animal cells and increase intracellular adenosine cyclic 3',5'-phosphate (cAMP) levels. We have purified the enzyme extensively from culture medium using anion-exchange chromatography in the presence and absence of calmodulin and gel filtration chromatography. The enzyme was purified 1600-fold to a specific activity of 608 mumol of cAMP min-1 mg-1 and was free of islet activating protein. The molecular weight of the enzyme was 43 400 in the absence of calmodulin and 54 200 in the presence of calmodulin. The Km of the bacterial enzyme for adenosine 5'-triphosphate was 2.0 mM, whereas the Km of the calmodulin-sensitive adenylate cyclase from bovine brain was 0.07 mM. Although the enzyme was not purified to homogeneity, its turnover number of 27 000 min-1 is the highest documented for any adenylate cyclase preparation.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.     

Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.     

Extracellular cAMP formation from host cell ATP by Bordetella pertussis adenylate cyclase

The effect of exogenously added adenylate cyclase from Bordetella pertussis (strain 114) has been investigated in Y-1 mouse adrenal tumor, chinese hamster ovary (CHO) and several other cells. A partially purified adenylate cyclase was found not to enter cells but, nevertheless, produced large amounts of cAMP in the medium. We could show that this resulted from release of ATP (and not larger molecules). The ATP released by the cells could be (1) directly measured and was replenished after each change of medium; (2) was reciprocally related to the cAMP produced; and (3) was competed for by ATPases present in added serum or by hexokinase and, less effectively, by exoenzymes on the cell surface. The extent of ATP leakage varied widely between different cell lines, being marked in CHO and Y-1 adrenal cells but negligible in transformed lymphocyte lines. The uncertainty of the origin of cAMP found in media of cultured cells requires separate analysis of cell and medium cAMP and an assessment of ATP leakage.     

Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera

Abstract To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli α-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with α-hemolysin.     

Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase

A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein (Mr = 46,659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.     

The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics.

The proportion of pyruvate dehydrogenase existing in the active form (PDHA) in suspensions of unstimulated cardiac myocytes oxidizing glucose is approx. 30%. Depolarization of the cells with concentrations of K+ above physiological values leads to an increase in the content of PDHA. Overloading of the cells with Na+ by treatment with veratridine and ouabain gives the same result. Each of these interventions is shown in experiments with Quin 2-loaded myocytes to lead to an increase in cytosolic free Ca2+ concentration ([Ca2+]c). Treatment of the cells with Ruthenium Red, an inhibitor of Ca2+ transport into mitochondria, largely prevents an increase in PDHA in response to addition of KCl or of veratridine plus ouabain. Ruthenium Red does not attenuate the increase in [Ca2+]c that occurs under these conditions. By contrast, treatment of the cells with ryanodine, an inhibitor of sarcoplasmic-reticulum Ca2+ transport and therefore of contraction, does not diminish the response of PDHA content to agents which raise [Ca2+]c; nor does loading of the cells with the Ca2+-chelating agent Quin 2, which also prevents contraction, at appropriate concentrations. It is concluded that an increase in [Ca2+]c causes an increase in PDHA content of cardiac myocytes independently of an increase in mechanical work. In the normal physiological situation the activation of dehydrogenases by Ca2+ is thought to help to maintain the balance of energy supply and demand during periods of increased work-load, which are associated with an increased myoplasmic [Ca2+]c.     

Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.     

The morphological changes in cultured cells caused by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis is the causative agent for human whooping cough. It was found that Bordetella pertussis infection caused a change in shape from flat to round in L2 cells, which are derived from rat type 2 alveolar cells. This phenomenon was reproduced using the culture supernatant of B. pertussis, and bacterium-free adenylate cyclase toxin (CyaA) was identified as the factor responsible. A purified preparation of wild-type CyaA but not an enzyme-dead mutant caused the cell rounding. It was examined whether CyaA causes similar morphological changes in various cultured cell lines. L2, EBL, HEK293T, MC3T3-E1, NIH 3T3, and Vero cells were rounded by the toxin whereas Caco-2, Eph4, and MDCK cells were not, although all these cells showed a significant elevation of the intracellular cAMP level in response to CyaA treatment, which indicates that there is no quantitative correlation between the rounding phenotype and the intracellular cAMP level. CyaA has been believed to target various immunocompetent cells and support the establishment of the bacterial infection by subverting the host immune responses. The possibility that CyaA may also affect tissue cells such as respiratory epithelial cells and may be involved in the pathogenesis of the bacterial infection is also indicated.     

Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase

The structural organization of the low molecular mass form (43 kDa) of Bordetella pertussis adenylate cyclase was dissected taking advantage of the known sequence of the bacterial cya gene (Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2, 19-30) and its low content of Trp and Met residues. Cleavage of the 43-kDa protein and of its complementary tryptic fragments (T25 and T18 peptides) with N-chlorosuccinimide and cyanogen bromide followed by sodium dodecyl sulfate-polyacrylamide gel analysis of digestion products allowed the following conclusions: (i) the catalytically active 43-kDa form of B. pertussis adenylate cyclase is within the first 400 residues of the protein encoded by the cya gene. T25 occupies the N-terminal domain of the protein (residues 1-235/237). Isolated T25 fragment exhibits a low but measurable enzymatic activity which indicates that it harbors the catalytic site; (ii) T18 which is the main calmodulin-binding domain, occupies the C-terminal segment of protein (residues 236/238-399) and is devoid of catalytic properties; (iii) the two complementary peptides T25 and T18 reassociated only in the presence of calmodulin, leading to significant recovery of the original activity. These results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.